Vaccinia virus has a genome of 185,000 bp that encodes approximately 200 polypeptides. These genes are expressed in a coordinated fashion so that some polypeptides are made before and others after DNA replication. In order to learn more about regulation of late gene expression, we sequenced an 8,600 base pair region of the vaccinia genome. Seven complete open-reading-frames were found. Six transcripts that initiated within this region were detected and of these four were synthesized only at late times. In each case, the transcriptional and translational start sites mapped within a few nucleotides of each other and the sequence TAAATG occurred at the start of the open-reading-frame. The putative promoter region of one late gene was analyzed by linking DNA segments to the coding region of the prokaryotic enzyme chloramphenicol acetyltransferase and recombining this construct into the vaccinia virus genome. By repeating this procedure with smaller and smaller putative promoter segments and assaying for enzyme activity, the location of the regulatory signals was determined. A rapidly sedimenting complex that accurately initiates and terminates transcription of early genes was isolated from vaccinia virus particles. Both a DNA-dependent ATPase and capping enzyme were associated with the transcription complex.